Comparing the Impact of Environmental Factors During Very High Gravity Brewing Fermentations

The impact of the initial dissolved oxygen, fermentation temperature, wort concentration and yeast pitching rate on the major fermentation process responses were evaluated by full factorial design and statistical analysis by JMP 5.01 (SAS software) software. Fermentation trials were carried out in 2L‐EBC tall tubes using an industrial lager brewing yeast strain. The yeast viability, ethanol production, apparent extract and real degree of fermentation were monitored. The results obtained demonstrate that very high gravity worts at 22°P can be fermented in the same period of time as a 15°P wort, by raising the temperature to 18°C, the oxygen level to about 22 ppm, and increasing the pitching rate to 22 × 10⁶ cell/mL. When diluting to obtain an 11.5°P beer extract, the volumetric brewing capacity increased 91% for the 22°P wort fermentation and 30% using the 15°P wort. After dilution, the fermentation of the 22°P wort resulted in a beer with higher esters levels, primarily the compound ethyl acetate.     

High Gravity Brewing by Continuous Process Using Immobilised Yeast: Effect of Wort Original Gravity on Fermentation Performance

The present work evaluated the influence of all‐malt wort original gravity on fermentative parameters and flavour‐active compound formation during primary fermentation of high gravity brewing by a continuous process using a lager yeast immobilised on a natural carrier obtained from brewer's spent grain (the main brewery by‐product). The all‐malt worts with original gravity (OG) ranging from 13.4 to 18.5°Plato were prepared by diluting a very‐high‐gravity wort (20°Plato) with sterile brewery water. The continuous assay was carried out in a bubble column bioreactor with a total working volume of 5.2 litres, at 15°C, using a constant gas flow rate of 250 mL/min (200 mL/min of CO₂ and 50 mL/min of air) and a dilution rate of 0.04 h^?1 (residence time of 25 h). The results indicated that as the wort OG was increased, the ethanol concentration of the outflowing beer increased. On the other hand, the continuous fermentation of the most concentrated worts (16.6 and 18.5°Plato) resulted in beers with unbalanced flavour profiles due to excessive ethyl acetate formation. The immobilised cell concentration appeared to be nearly independent from increasing wort OG.     

The Yeast Vacuole ‐A Scanning Electron Microscopy Study During High Gravity Wort Fermentations

The yeast vacuole has been shown to exhibit morphological responses to environmental conditions when exposed to worts of different gravity during fermentation. Marked effects of high gravity wort (20° Plato) on yeast morphology compared to more conventional wort gravity (12° Plato) were observed. High gravity worts caused vacuolar enlargement compared to conventional gravity wort. These results suggested that yeast cells experienced severe alterations with the vacuolar tonoplast when exposed to high osmotic pressure and elevated levels of ethanol.     

Fed‐batch fermentation with glucose syrup as an adjunct for high‐ethanol beer brewing

To produce a beer with a high ethanol content, preliminary research on fed‐batch fermentation profiles with glucose syrup as an adjunct during the primary fermentation period was conducted. The ethanol concentration of the beer was elevated by feeding a glucose syrup into the fermentors at a later stage of primary fermentation. Fermentation trials were carried out using a typical lager strain, SC‐9, with a pitching rate at 7.0 × 10⁶ cells/mL. An all‐malt wort (12.5°P) was employed and the primary fermentation temperature was 14 °C. Glucose syrup was supplemented when the concentration of residual reducing sugars was decreased to ~10 g/L. Results showed that the supplemented glucose was consumed rapidly and that the ethanol concentration in the final beer was raised to 67.9 g/L. Additional growth of yeast was observed after feeding accompanied by a low yield of ethanol (~0.46 g/g). Formation of diacetyl was enhanced by yeast growth and two additional peaks were obtained after feeding. The peak value of the diacetyl concentration was 1.90 mg/L. The fed‐batch fermentation resulted in a beer with an overproduction of higher alcohols and esters, indicating that brewing under these experimental conditions led to an unbalanced flavour profile. Results of optimization demonstrated that the optimal conditions were found to be 15°P for initial wort extract, 10 °C for fermentation temperature and 20 × 10⁶ cells/mL for yeast pitching rate, leading to total higher alcohols of 173.8 mg/L, total esters of 22.8 mg/L and an acetaldehyde concentration of 40.5 mg/L. A 12 day maturation and fermentation temperature of 8 °C was needed to reduce the acetaldehyde to 14.3 mg/L. Copyright © 2014 The Institute of Brewing & Distilling     

High gravity primary continuous beer fermentation using flocculent yeast biomass

The current work assessed a new immobilized cell reactor system throughout a long‐term (54 days) continuous primary fermentation of lager‐type wort of high specific gravity. The experiment was performed in a 4 L airlift bioreactor and immobilization of biomass was attained solely by flocculation. Despite the constant liquid agitation and washout of biomass, up to 53 g dry wt/L of yeast remained immobilized in the system. Two types of beer were produced without interrupting the reactor, based on two types of wort: a Pilsener type with high specific gravity of 15.6 ± 0.3°P; and a dark lager wort with specific gravity of 14.4 ± 0.03°P. Even during the inlet of high gravity wort, the desired attenuation was achieved without the need for either recirculation or an auxiliary second stage bioreactor. The specific saccharide consumption rate was kept around 7.9 ± 0.4 g/L/h and ethanol productivity oscillated at 3.36 ± 0.2 g/L/h for nearly a month. During this period the volumetric productivity of the current bioreactor reached 1.6 L beer/L/day. The green beers produced from the Pilsener and dark lager worts met the standards of regular finished primary beer fermentation. The productivity of diacetyl through the entire experiment could be correlated to the free amino nitrogen consumption rate. Copyright © 2014 The Institute of Brewing & Distilling     

INFLUENCE OF NITRATE AND BACTERIAL CONTAMINATION ON THE FORMATION OF APPARENT TOTAL N-NITROSO COMPOUNDS (ATNC) DURING FERMENTATION

Formation of Apparent Total N-Nitroso Compounds (ATNC) was monitored throughout fermentations of all-malt ale worts supplemented with nitrate (0–100 mg Litre^?1 (0–100 ppm)). The pitching yeasts were obtained from commercial breweries and contained different levels of the contaminant bacterium Obesumbacterium proteus (0–2.1% by number). Levels of ATNC present at the end of fermentation were dependent on both initial wort nitrate levels and the initial level of bacterial contamination of the pitching yeast. Only relatively low nitrate levels were required to produce ATNC levels greater than the Brewers' Society recommended limit of 20 μg Litre^?1 (ppb), provided that the bacteria were present. Indeed, the use of whole hops alone would contribute sufficient nitrate to the wort to produce excessive amounts of ATNC, provided O.proteus was present. The only feasible solution to ATNC production during fermentation is to remove the contaminating bacteria from both the pitching yeast and brewing plant. Effective removal of O.proteus can be achieved by acid washing the pitching yeast under carefully controlled conditions, prior to fermentation.     

THE EFFECT OF YEAST TREHALOSE CONTENT AT PITCHING ON FERMENTATION PERFORMANCE DURING BREWING FERMENTATIONS

The effect of yeast trehalose content at pitching on the fermentation performance during brewing fermentations was studied using a commercial strain of lager yeast, Saccharomyces cerevisiae (AJL 2155). Pitching yeasts with different trehalose contents were obtained by collecting cells in suspension after 96 h and 144 h of fermentation in EBC tubes in 10.8°P brewers wort at 14°C. The trehalose content of the pitching yeast had no effect on growth, specific gravity and ethanol production during the subsequent fermentation. A high trehalose content of the pitching yeast, however, sustained cell viability during the initial stage of fermentation, increased the carbohydrate utilisation rate and increased the production of isoamyl alcohol and isobutanol. For these aspects of fermentation performance, the trehalose content of the pitching yeast may prove useful in evaluating the vitality of pitching yeasts within the brewery .     

FERMENTATION OF WORTS MADE FROM 100% RAW SORGHUM AND ENZYMES

Worts made from raw sorghum and enzymes were successfully fermented even though the level of FAN present (51 mg/l) is well below that essential for fermentation of wort made from malted barley. Changes in typical fermentation parameters such as specific gravity, pH uptake of free amino nitrogen (FAN) and ammonium ions mirrored the increase in yeast cell concentration. Yeast viability remained high throughout the fermentation. Under identical fermentation conditions, malted barley worts showed typical fermentation profiles. However, malted barley worts with specific gravity maintained by the addition of D-glucose, but in which the FAN was diluted to a level similar to that found in a wort made from sorghum and enzymes, fermented more slowly and failed to attenuate fully. Five consecutive fermentations, using yeast cropped from the preceding to pitch the current fermentation were conducted. The specific gravity profiles were essentially the same in all five fermentations. Final values of pH, yeast in suspension, yeast viability and FAN were also indistinguishable. The yeast crop taken from fermentations of worts made from raw sorghum and enzymes represented a 5-fold increase over the initial pitching rate. When compared to commercial beers, the beers derived from fermentation of worts made from raw sorghum and enzymes contained lower levels of ethyl acetate, and higher levels of both 2- and 3-methyl butanol. In the beers derived from sorghum, isobutanol was always less than 20% of the total higher alcohol concentration.     

The Selection of a Dried Yeast Strain for Use in the Apparent Attenuation Limit Malt Analysis (AAL) Procedure

This investigation identifies that Mauribrew Lager 497 strain of dried yeast can be used as a standard strain for the determination of malt apparent attenuation limit (AAL). It provides ferment‐ability results for malt quality evaluation laboratories that are comparable to fresh brewery yeast. It was found that the optimal pitching rate in Congress wort (EBC Analytica, 1998, method 4.5.1), was 1 g per 200 mL, pitched at 25°C and fermented for 24 h at 20°C with agitation to complete attenuation. Preliminary trials also indicated that the Mauribrew Lager 497 dry yeast may be useful to brewers for determining the wort batch attenuation characteristics by the limit gravity test. In this case a pitching temperature of 35°C was found to be optimal with all other conditions as above. For the purpose of malt quality evaluation and brewery quality control the advantages of using a standard dry yeast strain include ease and convenience of use, consistency of quality, and uniformity between laboratories when they are located in separate geographic regions.     

Acid Washing and Serial Repitching a Brewing Ale Strain of Saccharomyces cerevisiae in High Gravity Wort and the Role of Wort Oxygenation Conditions†

Acid washing pitching yeast is an effective method for removing bacterial contamination, but if the yeast is washed incorrectly decreased fermentation performance and beer quality problems may result. Various factors can affect the acid resistance of yeast strains during brewery fermentations. Yeast from shaking flask experiments was more resistant to the combination of high gravity and acid washing conditions than yeast cropped from static fermentations. Yeast harvested from static high gravity wort (20° Plato; 1.083 OG) fermentations was more adversely affected by acid washing than yeast from standard gravity (12° Plato; 1.048 OG) wort. Wort oxygenation resulted in enhanced yeast fermentation performance and healthier yeast crops when yeast was serially repitched into 20° Plato wort. Yeast cropped from fermentations with air saturated high gravity wort responded poorly when acid washed. These results suggest that the structure of the plasma membrane particularly the sterol and fatty acid composition, may have an important role in tolerating high gravity wort and acid washing conditions.     

Characteristics of High Cell Density Fermentations with Different Lager Yeast Strains

To improve the productivity of the beer fermentation process, several strategies can be adopted. One of these promising strategies could be the increase of suspended yeast cells in the reactor. Therefore, the fermentation characteristics of 11 lager yeast strains were studied in normal pitched worts (20 × 10⁶ cells/ mL) (LD) and in worts with a four‐fold higher pitching rate (HD). The fermentation rate was 2–4 times increased when high initial cell levels were used. The net yeast growth was somewhat similar between the LD and the HD fermentations, although the FAN uptake level was about 35% higher in the HD fermentations compared with LD. High viabilities were observed throughout the fermentations with high cell loadings. HD fermentations resulted in higher concentrations of all the measured fusel alcohols and higher maxima and residual concentrations of total diacetyl were observed. In contrast, higher levels of most of the esters were found at the normal pitching rate, although the results of isoamyl acetate were not significant. With the help of “Principal Component Analysis”, it became clear that the cell density had an important influence on the flavour profile, but that yeast specific preferences could not be overlooked as they determined the sensitivity of the yeast to the application of higher cell densities.     

The effect of pitching rate on fermentation,maturation and flavour compounds of beer produced on an industrial scale

The aim of the study was to determine the effect of the initial number of yeast cells in the wort on the process of fermentation, maturation and the content of the volatile components of beer, as well as the viability and vitality of the yeast biomass. The experiments were performed on an industrial scale, with fermentation and maturation in cylindro‐conical fermentation tanks with a capacity of 3800 hL. Yeast for pitching was collected after secondary fermentation (third passage) and wort pitching levels were 5 × 10⁶, 7 × 10⁶ and 9 × 10⁶ cells/mL. During fermentation and maturation, the changes in the content of the extract, yeast growth, yeast vitality and selected volatile components were investigated. Experiments showed that the yeast inoculum had a significant impact on the course of the fermentation and metabolic changes. With increasing numbers of cells introduced into the wort, the content of the esters and fusel alcohols increased, while the acetaldehyde concentration decreased. These changes affected the final quality of the beer. Copyright © 2015 The Institute of Brewing & Distilling     

Maltose Transport by Brewer's Yeasts in Brewer's Wort

The kinetics of maltose transport by two industrial yeasts were studied. The ale and lager strain each showed both high and low affinity transport. For the lager strain, maltose transport was only weakly inhibited by maltotriose, sucrose and trehalose, suggesting that its dominant maltose transporter is the maltose‐specific type coded by MALx1 genes. For the ale strain, maltose transport was strongly inhibited by maltotriose, sucrose and trehalose, suggesting that its dominant maltose transporter may be the AGT1‐encoded type that also carries these sugars. Also glucose inhibited transport by the ale strain more than that by the lager strain. Instantaneous inhibition by ethanol at concentrations met in brewery fermentations was moderate (about 25% at 50 g ethanol · L^?1). The apparent Vmax for high affinity transport increased about 100‐fold between 0 and 30°C, whereas the Km (3 ± 1 mM) was constant. Standard activities of maltose transport and maltase were followed through pilot fermentations of 11–24°P worts. Rapid (20 s) measurements of the zero‐trans‐rate of maltose uptake were also made with each day's yeast (rapidly harvested and washed) in reaction mixtures containing the same day's wort labelled with tracer ¹⁴C‐maltose. Results suggested that maltose uptake is the dominant factor controlling the rate of maltose utilization in these wort fermentations.     

CAMBRIDGE PRIZE LECTURE IMPROVING YEAST FERMENTATION PERFORMANCE

A number of factors affecting yeast fermentation performance have been investigated. These include the mode of substrate feeding, nutrient supplementation, temperature, osmotic pressure, oxygen, intracellular ethanol accumulation, and yeast ethanol tolerance. Nutrient supplementation was observed to play a key role in yeast fermentations employing high gravity media and at high temperatures. Furthermore, complete attenuation of high gravity wort (25°P) could be achieved by optimizing the yeast pitching rate, fermentation temperature, and level of wort oxygenation. Genetic manipulation techniques, such as spheroplast fusion, were successfully employed to obtain ethanol and osmotolerant yeast strains. In addition, a number of stable 2-deoxy-D-glucose resistant mutants, isolated from brewing and non-brewing yeast strains, were observed to rapidly utilize maltose and maltotriose in the presence of high concentrations of glucose. Fermentation and ethanol production rates were increased in these strains. Therefore, employing strategies of optimized fermentation conditions and strain development have resulted in improvements in yeast fermentation performance.     

THE EFFECTS OF GLUCOSE ADJUNCT IN HIGH GRAVITY FERMENTATION BY SACCHAROMYCES CEREVISIAE 2036

Laboratory fermentations of 16°Plato glucose adjunct worts by Saccharomyces cerevisiae 2036 demonstrated the absence of “glucose repression” of maltose and maltotriose uptake. However, when compared to worts in which maltose syrup was employed as an adjunct, residual glucose was present at the end of fermentation, maltose and maltotriose uptake rates were enhanced, fructose uptake was blocked and the sequence of sugar uptake was changed. These findings partially explain residual glucose and fructose that sporadically appear in commercial beers. Further research suggests that the physiological quality of the yeast is of prime importance in carbohydrate metabolism, and that critical concentrations of glucose vary with different physiological conditions for this brewing strain in 16°P wort .     

THE PROPERTIES,COMPOSITION AND FERMENTABILITIES OF WORTS MADE FROM 100% RAW SORGHUM AND COMMERCIAL ENZYMES

A mashing regime was developed using 100% raw sorghum which enabled commercially acceptable hot water extracts to be obtained in 85 minutes with minimal use of a heat stable α-amylase and proteolytic enzymes. This gave worts of HWE 295 1°/kg, with FAN levels of about 40 mg/l and ammonium ion concentration of about 60 mg/l. Higher, but commercially unacceptable, levels of proteolytic enzymes gave worts with FAN from 84.5 to 95 (mg/l). Addition of an amyloglucosidase as the commercial preparation Amylo300L, was required to convert the HWE to fermentable extract. The addition of Amylo300L, increased the DP1, DP2 and DP3 carbohydrate fractions of the worts from 22% to more than 90% of the total, compared to about 80% for a wort made from malted barley without the use of enzymes. Two different proteolytic enzymes gave different extracts and FAN contents presumably reflecting either differences in susceptibilities of the sorghum to the two enzymes or the presence of different additional enzyme activities in the different preparations. The level of ammonium ions in malted barley worts was 86 mg/l and up to 88 mg/l in worts produced from sorghum and enzymes. Enzyme addition produced increased levels of ammonia. The content of Group A (the most readily assimilated) amino acids was proportionally higher in sorghum worts compared to malted barley wort. Worts made from raw sorghum and enzymes, containing as little as 40 mg/l FAN, were fully attenuated. The yeast consumed about 35 mg/l FAN and 45 mg/l ammonium ions. Under identical fermentation conditions, the same yeast, fermenting a malted barley wort of comparable extract consumed 104 mg/l FAN and 37 mg/l ammonium ions.     

Effect of Amyloglucosidase on Wort Composition and Fermentable Carbohydrate Depletion in Sorghum Lager Beers

A three‐factorial experiment with a level of confidence of P < 0.05 was performed to study fermentable carbohydrate depletion and ethanol production during 144 h fermentations of lager beers produced with barley malt (BM), sorghum malt (SM), refined maize (MZ) or waxy sorghum (WXSOR) grits treated during mashing with or without amyloglucosidase (AMG). The percentage glucose, maltose and maltotriose, based on total fermentable carbohydrates for the BM wort was 20, 68 and 13% and for the SM wort 35, 48 and 17% respectively. Treatment with AMG increased wort glucose from 9.3 to 24.5 g/L wort and total fermentable sugar equivalents, expressed as g glucose/L, from 59.2 to 72.6 g/L wort. The SM worts had approximately 50% more glucose and 40% less initial maltose content respectively compared to the BM worts. The WXSOR grits produced worts and beers with similar properties to those produced from the MZ adjuncts. AMG addition led to a >2.5 fold increment in wort glucose and 23% in total fermentable carbohydrate content. Linear regression analysis determined that the consumption rate of fermentable carbohydrates during fermentation followed first order reaction kinetics. Depletion times to reach 50% of the initial concentrations of glucose, maltose and maltotriose were 49, 128 and 125 h, respectively, clearly indicating that the fermenting yeast preferred glucose. Maltose and maltotriose depletion times of the AMG treated worts were significantly faster and lower, respectively, when compared with the untreated worts. At the end of the fermentation, the BM beers contained higher ethanol levels (5.1% v/v) than the SM beers (3.9% v/v). For AMG treated beers, no significant differences in ethanol content were observed among samples mashed with BM and beers produced from SM and MZ grits. The results demonstrated that AMG could be used to increase the initial concentration of glucose and total fermentable carbohydrates thus decreasing dextrin levels, especially from sorghum mashes.     

Degradation of Iso‐α‐Acids During Wort Boiling

A detailed study on the degradation of iso‐α‐acids was conducted. Because of the complexity of the wort matrix and interfering interactions during real wort boiling, the investigation of degradation kinetics was performed in an aqueous solution. Degradation was investigated as a function of time (0–90 min), temperature (80–110°C), pH value (4–7), original gravity (10–18°P) and ion content of the water (0–500 ppm Ca²⁺ and Mg²⁺). After 90 min of boiling, over 20% of the dosed iso‐α‐acids could no longer be detected. A strong dependence of degradation could be shown due to high temperature, low pH, high original gravity and a high Mg²⁺ content. The cis:trans ratio and co‐iso‐α‐acid content did not change significantly. Losses of isohumulones could be lowered by reducing the temperature and original gravity, as well as by heightening the pH value. High amounts of Ca²⁺ and Mg²⁺ salts also led to an increase in degradation products. Solutions to decrease degradation and thereby possible improvements in sensory bitter quality are discussed.     

HYDROPHOBIC POLYPEPTIDE EXTRACTION DURING HIGH GRAVITY MASHING— EXPERIMENTAL APPROACHES FOR ITS IMPROVEMENT

The aim was to establish if a substantial increase in hydrophobic polypeptides could be achieved during high gravity mashing. When worts with gravities ranging from 5–20°P were analysed for hydrophobic polypeptide content it was found that there was no appreciable increase in hydrophobic polypeptide levels. Remashing of the spent grains from low and high gravity mashes demonstrated that this resulted from inefficient extraction of hydrophobic polypeptide levels during the mashing process. For example, wort produced from remashed high gravity spent grains contained 150 mg/L hydrophobic polypeptides compared to only 10 mg/L in the low gravity remashed spent grains. Experiments were conducted, employing standard mashing techniques, in an attempt to increase the extraction of hydrophobic polypeptides during high gravity mashing. Thus the use of gypsum, proteolytic stands, varying liquor to grist ratios and wheat malt addition were all investigated for their effect on hydrophobic polypeptide extraction during high and low gravity mashing. Wort analysis demonstrated that none of the techniques employed had a significant effect on hydrophobic polypeptide extraction. When wort from remashed spent grains was used as mashing in liquor for a fresh mash and the resultant worts analysed for hydrophobic polypeptides it was observed that no increase in hydrophobic polypeptide extraction was achieved. For example, wort from the remashed high gravity spent grains, containing 140 mg/L hydrophobic polypeptides, when used as mashing-in liquor, produced no increase in hydrophobic polypeptide levels in the resultant high gravity wort (230 mg/L) when compared to a high gravity wort produced using distilled water as mashing-in liquor (255 mg/L). It is therefore concluded that a saturation point has been reached and no more hydrophobic polypeptides can be extracted during mashing regardless of the procedures employed.     

Optimised Acidification Power Test of Yeast Vitality and its Use in Brewing Practice

The optimised acidification power test (APT) of brewer's yeast quality includes storing the yeast slurry at 2°C under beer (AP remains constant for up to 6 days), a 15 min sample equilibration to room temperature, decantation, and washing by triple centrifugation in deionised water. The final yeast pellet keeps its AP for up to 6 h at room temperature under water and thus the APT does not need to be performed immediately after yeast collection. The correct AP value (maximum acidification produced by given yeast) is determined at 25 ± 0.1°C in a 15 mL sample containing ≥5% glucose and ≥1.5 g yeast wet weight. The cell concentration is conveniently measured as absorbance (A₆₆₀). Cell flocculation and/or sedimentation that can distort APT results can be prevented by stirring the sample at ≥200 rpm. The AP of yeast of different generations used to pitch brewery fermentations in cylindroconical tanks had a very low correlation with the wort half‐attenuation time (T_1/2) due to large scatter, while each yeast generation separately showed a clear T_1/2‐AP relationship. The lowest AP of yeast cropped from cylindroconical tanks was displayed by the first cropped fraction. Post‐cropping cooling had no effect on AP. Variations in pitching yeast vitality and their effect on the outcome of a brewery fermentation can be masked by variations in pitching rate, wort composition, ambient conditions in the cylindroconical tanks and other factors.